Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Turning a Drug Target into a Drug Candidate: A New Paradigm for Neurological Drug Discovery?

The conventional paradigm for developing new treatments for disease mainly involves either the discovery of new drug targets, or finding new, improved drugs for old targets. However, an ion channel found only in invertebrates offers the potential of a completely new paradigm in which an established drug target can be re-engineered to serve as a new candidate therapeutic agent. The L-glutamate-gated chloride channels (GluCls) of invertebrates are absent from vertebrate genomes, offering the opportunity to introduce this exogenous, inhibitory, L-glutamate receptor into vertebrate neuronal circuits either as a tool with which to study neural networks, or a candidate therapy. Epileptic seizures can involve L-glutamate-induced hyper-excitation and toxicity. Variant GluCls, with their inhibitory responses to L-glutamate, when engineered into human neurons, might counter the excitotoxic effects of excess L-glutamate. In reviewing recent studies on model organisms, it appears that this approach might offer a new paradigm for the development of candidate therapeutics for epilepsy.

Invertebrate neurones, genomes, phenotypic and target-based screening; their contributions to the search for new chemical leads and new molecular targets for the control of pests, parasites and disease vectors.

Insect-borne diseases of humans, animals and plants can be devastating. The direct damage to crops by insect and nematode pests can also severely reduce crop yields and threaten harvests. Parasitic nematodes can impair human health and the health of farm livestock. Effective control for all such pests, vectors and pathogens is required as the economic and health burden can be substantial. Insecticides, nematicides and anthelmintics have been at the forefront of control and will remain important in the immediate future, even as we explore new and more sustainable methods to maintain the necessary disease control and the growth in food supply. Many important chemicals deployed for the control of invertebrate disease vectors and pathogens of humans, agricultural crops and farm livestock are active on ion channels, resulting in rapid actions. Understanding their modes of action has been accelerated by studies on the physiology of identifiable invertebrate excitable cells. Nematode and insect genetic model organisms and comparative genomics have contributed to defining the molecular targets of insecticides and anthelmintics, facilitating target-based screening. Automated phenotyping, which allows high-throughput screening of chemical libraries for new and re-purposed compounds, has been increasingly deployed in the search for new molecules of interest. With a growing world population to be fed and a 20-49% loss of global harvest to pests, we need to maintain control of the pests, parasites and pathogens that threaten global food supply and global health.

Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti.

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe, and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50 of 25.5 ± 5.0 µM and 22.3 ± 5.4 µM, respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are inhibitors of topoisomerase I, have known activity against several agricultural pests, and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries.

Actions on mammalian and insect nicotinic acetylcholine receptors of harmonine-containing alkaloid extracts from the harlequin ladybird Harmonia axyridis.

The harlequin ladybird, Harmonia axyridis (H. axyridis), possesses a strong chemical defence that has contributed to its invasive success. Ladybird beetle defensive chemicals, secreted in response to stress and also found on the coating of laid eggs, are rich in alkaloids that are thought to be responsible for this beetle's toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists, hence we have explored the actions of alkaloids of the ladybird H. axyridis on both mammalian and insect nAChRs. Electrophysiological studies on native and functionally expressed recombinant nAChRs were used to establish whether an alkaloid extract from H. axyridis (HAE) targeted nAChRs and whether any selectivity exists for insect over mammalian receptors of this type. HAE was found to be an inhibitor of all nAChRs tested with the voltage-dependence of inhibition and the effect on ACh EC50 differing between nAChR subtypes. Our finding that an HAE fraction consisting almost entirely of harmonine had a strong inhibitory effect points to this alkaloid as a key component of nAChR inhibitory actions. Comparison of HAE inhibition between the mammalian and insect nAChRs investigated indicates some preference for the insect nAChR supporting the view that investigation of ladybird alkaloids shows promise as a method for identifying natural product leads for future insecticide development.

Anthelmintic drug discovery: target identification, screening methods and the role of open science.

Helminths, including cestodes, nematodes and trematodes, are a huge global health burden, infecting hundreds of millions of people. In many cases, existing drugs such as benzimidazoles, diethylcarbamazine, ivermectin and praziquantel are insufficiently efficacious, contraindicated in some populations, or at risk of the development of resistance, thereby impeding progress towards World Health Organization goals to control or eliminate these neglected tropical diseases. However, there has been limited recent progress in developing new drugs for these diseases due to lack of commercial attractiveness, leading to the introduction of novel, more efficient models for drug innovation that attempt to reduce the cost of research and development. Open science aims to achieve this by encouraging collaboration and the sharing of data and resources between organisations. In this review we discuss how open science has been applied to anthelmintic drug discovery. Open resources, including genomic information from many parasites, are enabling the identification of targets for new antiparasitic agents. Phenotypic screening remains important, and there has been much progress in open-source systems for compound screening with parasites, including motility assays but also high content assays with more detailed investigation of helminth physiology. Distributed open science compound screening programs, such as the Medicines for Malaria Venture Pathogen Box, have been successful at facilitating screening in diverse assays against many different parasite pathogens and models. Of the compounds identified so far in these screens, tolfenpyrad, a repurposed insecticide, shows significant promise and there has been much progress in creating more potent and selective derivatives. This work exemplifies how open science approaches can catalyse drug discovery against neglected diseases.

Combined effects of mutations in loop C and the loop D-E-G triangle on neonicotinoid interactions with Drosophila Dα1/chicken ß2 hybrid nAChRs.

Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken ß2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Dr. Kathleen Drew-Baker, "Mother of the Sea", a Manchester scientist celebrated each year for half a century in Japan.

2013 marks the 50th annual Drew festival in Uto City, Japan, celebrating the work of University of Manchester botanist, Dr. Kathleen Drew-Baker. Her insight into the reproductive biology of algae was the key to efficient farming of the seaweed "nori" which is a familiar component of Japanese food.

Probing new components (loop G and the α-α interface) of neonicotinoid binding sites on nicotinic acetylcholine receptors.

Neonicotinoid insecticides interact with the orthosteric site on the extracellular ligand binding domain (LBD) of nicotinic acetylcholine receptors (nAChRs), typically activating the cation permeable ion channels. In nAChRs consisting of two α and three non-α subunits, LBDs contain six loops (loops A, B and C on the α subunit and loops D, E and F on the non-α subunit) which make up the orthosteric binding site at the α/non-α subunit interfaces. Recently, an additional site (loop G) on the ß1 strand has been identified. Also, when the α/non-α subunit ratio is 3/2, another binding site is generated at the interface of two adjacent α subunits. Roles for loop G and the α-α interface in the interactions with neonicotinoids are discussed with reference to recent structural and physiological data.

Alzheimer's disease: insights from Drosophila melanogaster models.

The power of fruit fly genetics is being deployed against some of the most intractable and economically significant problems in modern medicine, the neurodegenerative diseases. Fly models of Alzheimer's disease can be exposed to the rich diversity of biological techniques that are available to the community and are providing new insights into disease mechanisms, and assisting in the identification of novel targets for therapy. Similar approaches might also help us to interpret the results of genome-wide association studies of human neurodegenerative diseases by allowing us to triage gene "hits" according to whether a candidate risk factor gene has a modifying effect on the disease phenotypes in fly model systems.

Exon 3 splicing and mutagenesis identify residues influencing cell surface density of heterologously expressed silkworm (Bombyx mori) glutamate-gated chloride channels.

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for l-glutamate and ivermectin (IVM); however, Imax (the maximum l-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest l-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on l-glutamate and IVM actions. Binding assays using [(3)H]IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced Bmax but not Kd values of [(3)H]IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the l-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response.

Studies on an acetylcholine binding protein identify a basic residue in loop G on the ß1 strand as a new structural determinant of neonicotinoid actions.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the ß1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the α7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.

An antagonist of the retinoid X receptor reduces the viability of Trichuris muris in vitro.

BACKGROUND: Trichuriasis is a parasitic disease caused by the human whipworm, Trichuris trichiura. It affects millions worldwide, particularly in the tropics. This nematode parasite burrows into the colonic epithelium resulting in inflammation and morbidity, especially in children. Current treatment relies mainly on general anthelmintics such as mebendazole but resistance to these drugs is increasingly problematic. Therefore, new treatments are urgently required. METHODS: The prospect of using the retinoid X receptor (RXR) antagonist HX531 as a novel anthelmintic was investigated by carrying out multiple viability assays with the mouse whipworm Trichuris muris. RESULTS: HX531 reduced both the motility and viability of T. muris at its L3, L4 and adult stages. Further, bioinformatic analyses show that the T. muris genome possesses an RXR-like receptor, a possible target for HX531. CONCLUSIONS: The study suggested that Trichuris-specific RXR antagonists may be a source of much-needed novel anthelmintic candidates for the treatment of trichuriasis. The identification of an RXR-like sequence in the T. muris genome also paves the way for further research based on this new anthelmintic lead compound.

A single amino acid polymorphism in the Drosophila melanogaster Dα1 (ALS) subunit enhances neonicotinoid efficacy at Dα1-chicken ß2 hybrid nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.

Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. Here we investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster Dα1 subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 µM ACh, of the Dα1-chicken ß2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of Dα1 with these neonicotinoids.

Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito.

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background in which there is not a clear orthologue in Vectorbase and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (6), chloropyriphos (6.6) and fenthion (3.2) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies.

A novel Caenorhabditis elegans allele, smn-1(cb131), mimicking a mild form of spinal muscular atrophy, provides a convenient drug screening platform highlighting new and pre-approved compounds.

Spinal muscular atrophy (SMA), an autosomal recessive genetic disorder, is characterized by the selective degeneration of lower motor neurons, leading to muscle atrophy and, in the most severe cases, paralysis and death. Deletions and point mutations cause reduced levels of the widely expressed survival motor neuron (SMN) protein, which has been implicated in a range of cellular processes. The mechanisms underlying disease pathogenesis are unclear, and there is no effective treatment. Several animal models have been developed to study SMN function including the nematode, Caenorhabditis elegans, in which a large deletion in the gene homologous to SMN, smn-1, results in neuromuscular dysfunction and larval lethality. Although useful, this null mutant, smn-1(ok355), is not well suited to drug screening. We report the isolation and characterization of smn-1(cb131), a novel allele encoding a substitution in a highly conserved residue of exon 2, resembling a point mutation found in a patient with type IIIb SMA. The smn-1(cb131) animals display milder yet similar defects when compared with the smn-1 null mutant. Using an automated phenotyping system, mutants were shown to swim slower than wild-type animals. This phenotype was used to screen a library of 1040 chemical compounds for drugs that ameliorate the defect, highlighting six for subsequent testing. 4-aminopyridine, gaboxadol hydrochloride and N-acetylneuraminic acid all rescued at least one aspect of smn-1 phenotypic dysfunction. These findings may assist in accelerating the development of drugs for the treatment of SMA.